Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2)

We report the preparation and structure–activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.     

Molecular phylogenetics and biogeography of the Neocellia Series of Anopheles mosquitoes in the Oriental Region

Molecular studies of population divergence and speciation across the Oriental Region are sparse, despite the region’s high biodiversity and extensive Pliocene and Pleistocene environmental change. A molecular phylogenetic study of the Neocellia Series of Anopheles mosquitoes was undertaken to identify patterns of diversification across the Oriental Region and to infer the role of Pleistocene and Pliocene climatic change. A robust phylogeny was constructed using CO2 and ND5 mitochondrial genes and ITS2 and D3 nuclear ribosomal markers. Bayesian analysis of mitochondrial genes was used to date divergence events. The repeated contraction and expansion of forest habitat resulting from Pleistocene climatic fluctuations appears to have had a substantial impact on intraspecific diversification, but has not driven speciation within this group. Primarily early to mid Pliocene speciation was detected within the Annularis Group, whereas speciation within the Maculatus and Jamesii Groups occurred during the mid and late Pliocene. Both allopatric divergence driven by late Pliocene environmental changes and ecological adaptation, involving altitudinal replacement and seasonality, are likely to have influenced speciation in the Maculatus Group.     

Plant small monomeric G-proteins (RAC/ROPs) of barley are common elements of susceptibility to fungal leaf pathogens,cell expansion and stomata development

Small monomeric RAC/ROP GTPases act as molecular switches in signal transduction processes of plant development and stress responses. They emerged as crucial players in plant-pathogen interactions either by supporting susceptibility or resistance. In a recent publication, we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs regulate susceptibility to barley fungal leaf pathogens of different life style in a contrasting way. This illustrates the distinctive signalling roles of RAC/ROPs for different plant-pathogen combinations. We also reported the involvement of RAC/ROPs in plant epidermis development in a monocotyledonous plant. Here we further discuss a failure of CA HvRAC/ROP-expressing barley to normally develop stomata.Key words: Hordeum vulgare, G-proteins, RAC, ROP, cell expansion, stomata, transpirationMembers of the RHO family of small G-proteins in plants (RAC/ROPs) regulate signal transduction processes at the plasma membrane.¹ They act as multifunctional signalling switches in plant development and a variety of stress responses. RAC/ROP GTPases play regulatory roles in polar growth and cell morphogenesis in several cell systems including pollen tubes, developing root hairs and leaf pavement cells.²In a recent publication,³ we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs support susceptibility to the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). CA HvRAC1 supported susceptibility to biotrophic Bgh but resistance to hemibiotrophic Magnaporthe oryzae in barley at the penetration level in both cases. Additionally, CA HvRAC1 supported local callose deposition at sites of attack from Bgh and a secondary H₂O₂ burst in whole non-penetrated epidermal cells. This supports a regulatory function of RAC/ROPs in plant defence¹ and the potential corruption of defence pathways in susceptibility to Bgh. Because the rice ortholog of HvRAC1, OsRAC1, can regulate an H₂O₂ burst via activation of the plasma membrane NADPH oxidase OsRBOHB,⁴ one can speculate that the secondary H₂O₂ burst in CA HvRAC1 barley could also be caused by over-activation of an NADPH oxidase. However, CA HvRAC1 barley was also more susceptible to fungal penetration, and penetrated cells did not show an H₂O₂ burst. Hence, CA HvRAC1 did not contribute to penetration resistance, and the H₂O₂ burst might have been suppressed by Bgh after successful penetration. Interestingly, Bgh secretes a catalase during interaction with the plant.⁵The involvement of RAC/ROPs in plant development has been widely studied in the dicots Arabidopsis and tobacco. In Arabidopsis, CA AtRAC/ROPs disturb root hair tip growth and epidermal cell morphogenesis.^6,7 We showed similar developmental aberrations as a result of CA HvRAC/ROP expression in monocotyledonous barley. Root hair polarity disruption and enhanced leaf epidermal cell expansion was observed in CA HvRAC/ROP expressing barley. Here, we further report on reduced or abnormal development of stomata as an effect of CA HvRAC/ROP expression.In barley, stomata and short epidermal cells alternate in a row of leaf epidermal cells (Fig. 1A). The number of stomata number was significantly reduced in three CA HvRAC/ROP (CA HvRACB, CAHvRAC3, CA HvRAC1) expressing barley genotypes when compared to azygous controls (barley siblings that lost the transgene due to segregation) (Fig. 1E). In part, this could be explained by enhanced length of epidermal cells intercalated between stomata (Fig. 1B). The presence of longer epidermal cells in all CA HvRAC/ROP-barleys further supports that RAC/ROPs are operating in epidermal cell expansion.³Open in a separate windowFigure 1Stomatal abnormalities observed in CA HvROPexpressing transgenic barley leaves. (A) Wild type leaf adaxial epidermis with alternating stomata complexes (arrows) and short epidermal cells (asterisk). (B) Presence of more than one short epidermal cell in between two stomata. Arrows point the stomata. Double headed arrows highlight intercalated cells with enhanced cell length (C) Two stomata lacking an intercalated short epidermal cell. (D) Stoma failed to develop and left an abnormal blank cell. (E) Average number of stomata present in 5 cm of a stomatal row in transgenic plants expressing distinct CA barley CA HvRAC/ROPs. For all samples, stomatal rows present on either side of the mid rib were counted in the leaf upper epidermis. Fully expanded leaves of 3-weeks-old barley plants were used for counting stomata. Error bars show 95% confidence intervals. Repetition of the experiment led to similar results. Scale bars = 50 µm.Previously, we carried out porometry experiments to measure stomata conductivity in CA HvRACB expressing barley leaves.⁸ The CA HvRACB leaves showed up to 50% less transpiration than azygous controls without any treatment. Additionally, CA HvRACB leaves were less responsive to abscisic acid (ABA) and subsequently they could not effectively reduce transpiration when treated with ABA or when cut-off from water supply.⁸ Our data on numbers of stomata per leaf segment could now explain the lower rates of transpiration in non-stressed CA HvRACB barley when compared to wild type.Apart from the stomata number, developmental abnormalities were observed in the arrangement of epidermal cells. Generally, the shape of epidermal cells was less regular in CA HvRAC/ROP barley.³ We also observed the presence of more than one short epidermal cell in between two stomata (Fig. 1B) or two stomata lacking an intercalated short epidermal cell (Fig. 1C), or stomata failed to develop, which ended up in an abnormally short epidermal cell (Fig. 1D). Although such abnormalities were also rarely observed in wild type plants, all three CA HvRAC/ROP-barley leaves exhibited a clearly higher frequency of abnormalities in a given length of a stomata row. Together, CA HvRAC/ROPs had an effect on both the number and development of stomata. These observations suggest that RAC/ROPs are not only operating in cell expansion but also in barley cell differentiation for stomata development.     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6 mm × 75 mm, 3.5 μm, 80 Å column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6 mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2 → 200.1 and 387.1 → 203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00–3000.00 ng/mL with correlation coefficient (r) ≥ 0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13–1.54 and 1.76–2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40 mg) in healthy human volunteers.     

Multiple mechanisms mediate motor neuron migration in the zebrafish hindbrain

The transmembrane protein Van gogh‐like 2 (Vangl2) is a component of the noncanonical Wnt/Planar Cell Polarity (PCP) signaling pathway, and is required for tangential migration of facial branchiomotor neurons (FBMNs) from rhombomere 4 (r4) to r5‐r7 in the vertebrate hindbrain. Since vangl2 is expressed throughout the zebrafish hindbrain, it might also regulate motor neuron migration in other rhombomeres. We tested this hypothesis by examining whether migration of motor neurons out of r2 following ectopic hoxb1b expression was affected in vangl2^? (trilobite) mutants. Hoxb1b specifies r4 identity, and when ectopically expressed transforms r2 to an “r4‐like” compartment. Using time‐lapse imaging, we show that GFP‐expressing motor neurons in the r2/r3 region of a hoxb1b‐overexpressing wild‐type embryo migrate along the anterior‐posterior (AP) axis. Furthermore, these cells express prickle1b (pk1b), a Wnt/PCP gene that is specifically expressed in FBMNs and is essential for their migration. Importantly, GFP‐expressing motor neurons in the r2/r3 region of hoxb1b‐overexpressing trilobite mutants and pk1b morphants often migrate, even though FBMNs in r4 of the same embryos fail to migrate longitudinally (tangentially) into r6 and r7. These observations suggest that tangentially migrating motor neurons in the anterior hindbrain (r1‐r3) can use mechanisms that are independent of vangl2 and pk1b functions. Interestingly, analysis of tri; val double mutants also suggests a role for vangl2‐independent factors in neuronal migration, since the valentino mutation partially suppresses the trilobite mutant migration defect. Together, the hoxb1b and val experiments suggest that multiple mechanisms regulate motor neuron migration along the AP axis of the zebrafish hindbrain. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

Kinetic modeling of tricarboxylic acid cycle and glyoxylate bypass in Mycobacterium tuberculosis, and its application to assessment of drug targets

Background  

Targeting persistent tubercule bacilli has become an important challenge in the development of anti-tuberculous drugs. As the glyoxylate bypass is essential for persistent bacilli, interference with it holds the potential for designing new antibacterial drugs. We have developed kinetic models of the tricarboxylic acid cycle and glyoxylate bypass in Escherichia coli and Mycobacterium tuberculosis, and studied the effects of inhibition of various enzymes in the M. tuberculosis model.     

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.     

Interaction of the disaccharide trehalose with a phospholipid bilayer: a molecular dynamics study

The disaccharide trehalose is well known for its bioprotective properties. Produced in large amounts during stress periods in the life of organisms able to survive potentially damaging conditions, trehalose plays its protective role by stabilizing biostructures such as proteins and lipid membranes. In this study, molecular dynamics simulations are used to investigate the interaction of trehalose with a phospholipid bilayer at atomistic resolution. Simulations of the bilayer in the absence and in the presence of trehalose at two different concentrations (1 or 2 molal) are carried out at 325 K and 475 K. The results show that trehalose is able to minimize the disruptive effect of the elevated temperature and stabilize the bilayer structure. At both temperature, trehalose is found to interact directly with the bilayer through hydrogen bonds. However, the water molecules at the bilayer surface are not completely replaced. At high temperature, the protective effect of trehalose is correlated with a significant increase in the number of trehalose-bilayer hydrogen bonds, predominantly through an increase in the number of trehalose molecules bridging three or more lipid molecules.